The present invention generally relates to the production of 2-keto-L-gulonic acid by microbial (fermentative or enzymatic) reduction of 2,5-diketo-D-gluconic acid. Particularly, it is concerned with (i) use of a newly induced mutant defective in metabolizing 5-keto-D-gluconic acid or its processed product in the reduction process, (ii) the mutant itself, used in performing the process, (iii) addition of a nitrate salt and a hydrogen donor to the reduction system, and (iv) sterilization with a surfactant of a fermentation broth in which a 2,5-diketo-D-gluconic acid producing microorganism has been cultured and which is used as the raw material to be reduced by the mutant or its processed product.